Capillary electrophoretic separation techniques find wide application in the biologically related sciences. Molecular species such as peptides, proteins, oligonucleotides, and oligosaccharides are separated by causing them to migrate in a buffer solution under the influence of an electric field. The separation is normally carried out in thin-walled, narrow-bore capillary tubes to minimize the evolution of heat during electrophoretic separation, which would cause zone deformation.
Among the other mechanisms that can cause zone deformation are non-uniform electroendosmosis, excess electroosmotic flow, and solute adsorption to the inner surface of the capillary. However, these problems can be minimized or overcome by coating the inner wall of the electrophoresis tube with various polymeric substances.
In U.S. Pat. No. 4,680,201, Hjerten discloses a method for coating the inner wall of a narrow bore capillary with a monomolecular polymeric coating of polyacrylamide bonded to the capillary wall by means of a bifunctional reagent, e.g., xcex3-methacryloxypropyltrimethoxysilane. These capillaries can be used for free-zone electrophoresis in open tubes.
Novotny et al., U.S. Pat. No. 5,074,982, discloses that the inner wall of silica capillaries used in electrophoretic separations can be coated with bifunctional reagent using a Grignard reagent, for hydrolytic stability.
Thermal immobilization of adsorbed polyvinyl alcohol (PVA) as a coating on fused silica capillary surfaces is described in Gilges et al., Anal. Chem. 66:2038-2046 (1994). These coatings are stable for separations over a wide range of pH; however, at high buffer pH, the adsorption of PVA molecules and the suppression of analyte/wall interaction is weakened.
The present invention generally features coatings suitable for surfaces such as are found in capillary electrophoresis columns and methods for their preparation. A microcapillary column of the invention generally includes a microcapillary having an interior cavity and a wall with an inner surface, the inner surface of the wall having an interconnected polymeric coating that includes a functional group attached to the inner surface and capable of copolymerizing with an organic compound in an organic solvent and a polymer of the organic compound copolymerized with the functional group. The coating can be covalently or non-covalently attached to the column wall and can further include an additional layer of coating material. Preferably, the organic compound is a vinyl ester, and most preferably, vinyl acetate, and the attached polymer forming the exposed surface of the coating is a polyvinyl alcohol, the hydroxyl groups of which can be further derivatized in any desired manner. In a most preferred capillary column, the coating material includes a polyvinyl alcohol based polymer covalently attached to the column wall by Sixe2x80x94Oxe2x80x94Si bonds.
The method of the invention generally includes providing a microcapillary, modifying the inner surface of the capillary wall to provide attached functional groups capable of copolymerizing with an organic compound in an organic solvent, introducing a solution of the organic compound in an organic solvent into the interior cavity of the microcapillary; and causing molecules of the organic compound to copolymerize with the attached functional groups to form an interconnected polymeric coating material attached to the inner surface of the microcapillary column. Preferably, the attached functional groups are covalently bonded vinyl groups, the organic compound is a vinyl ester, and the resulting interconnected polymeric coating material of polyvinyl ester is modified in a polymer homologous reaction to produce the desired polyvinyl alcohol coating.
In another aspect, the method of the invention features, in general, forming a column with a hydrophilic polymeric coating by directly converting an attached hydrophobic polymeric coating material to a hydrophilic coating material. The resulting hydrophobic polymeric coating can contain acidic, basic or neutral functionalities depending on the intended use of the column.
The terms xe2x80x9cCE columnxe2x80x9d or xe2x80x9cmicrocapillary columnxe2x80x9d are meant to include a vessel of any shape in which capillary electrophoresis can be carried out. For example, it is also known to use chips with open grooves microfabricated into the surface of the chip for capillary electrophoresis.
The coating of the invention creates a new, stable surface, appropriate for CE columns or general surface modification. The coating is stable over a wide pH range and allows highly efficient grafting and/or adsorption of a variety of additional layers, if desired. As used in capillary electrophoresis, the coating suppresses or controls electroosmotic flow and prevents adsorption of analytes to the surface of the column.